The Ras family of proteins and several of the nuclear lamins contain a carboxyl-terminal CaaX motif that triggers protein isoprenylation and methylation. We have intensively studied these posttranslational modifications. We created gene-targeted mice to examine the role of all of the enzymes involved in the posttranslational processing of CaaX proteins [protein farnesyltransferase (FTase), protein geranylgeranyl- transferase-I (GGTase-I), ICMT, RCE1, ZMPSTE24]. We have examined the importance of each enzyme in the growth of Ras-induced tumors. We also discovered that ZMPSTE24 is a prelamin A protease and that its absence leads to an accumulation of farnesyl-prelamin A. This accumulation of farnesyl-prelamin A underlies certain progeroid disorders in humans, including Hutchinson-Gilford progeria syndrome. We created gene- targeted mice and investigated the importance of protein farnesylation in the pathogenesis and treatment of progeria. We also discovered that lamins B1 and B2 are crucial for neuronal migration in the brain. Our research program on CaaX proteins has been productive, resulting in 38 publications during the past 5 years. The objective of this renewal application is to continue our work on the in vivo importance of the modifications of CaaX proteins, focusing on specific topics relevant to human disease. Specific Aim 1 is to use genetically modified mice to examine the importance of lamins B1 and B2 in the brain and in other tissues. Our preliminary studies suggest that B-type lamins are essential for neuronal migration. We need to investigate this issue in depth. Also, we need to determine whether the individual proteins, lamin B1 and lamin B2, have intrinsically different roles in this process. Finally, we need to investigate the roles of lamin B1 and lamin B2 in adult tissues. The fact that lamin B1 and lamin B2 knockout mice die at birth means that no one has yet explored the physiologic importance of these proteins in adult tissues. Very recently, we have created conditional knockout alleles for both Lmnb1 and Lmnb2, and over the next few years, we intend to use these tools to address the importance of the B-type lamins in adult tissues. Specific Aim 2 is to define the importance of the posttranslational modifications of prelamin A in the assembly of the nuclear lamina, and to define the importance of protein farnesylation in the pathogenesis of progeroid syndromes. Over the past few years, our laboratory has generated knock-in mice that synthesize exclusively prelamin A and other knock-in mice that produce exclusively mature lamin A (where lamin A is produced directly, bypassing the posttranslational processing). Already, we have discovered differences in the assembly of the nuclear lamina in these mice. Further analyses of these mice should help us to define the physiologic importance of the posttranslational processing of prelamin A. Also, additional knock-in mice created by our group have strongly suggested that the farnesyl lipid modification may be critical for certain progeroid disorders, but less critical for others. We plan to explore the cell biology underlying this finding. PUBLIC HEALTH RELEVANCE: The proteins of the nuclear lamina have generated enormous interest because of recent studies showing that genetic defects in nuclear lamins cause multiple human diseases. The main objective of this renewal application is to continue to explore the physiologic and medical relevance of the posttranslational processing of the nuclear lamins, particularly as it relates to devastating neuronal migration defects and the pathogenesis and treatment of progeroid syndromes (precocious aging syndromes). Our studies will be focused on the physiologic importance of the B-type lamins in the developing brain and other tissues and the physiologic importance of the posttranslational processing of prelamin A.